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bioRxiv · 10.1101/2025.11.13.688285

Construction of a CMV promoter-driven Langat virus cDNA clone and reporter viruses

Hermacinski, A. I.·Richardson, R. B.·Bednarski, E.·Salim, M.·Garg, A.·Lim, J. K.·Evans, M. J.
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Abstract

Langat virus (LGTV) is a tick-borne member of the Flaviviridae family and a biosafety level 2 surrogate for studying tick-borne encephalitis virus (TBEV) replication and pathogenesis. Here, we report the construction of a plasmid encoding a cytomegalovirus (CMV) promoter-driven LGTV cDNA that initiates infection following direct transfection of mammalian cells. Incorporation of three introns eliminated viral cDNA-associated toxicity in bacteria, enabling stable propagation of the full-length plasmid. Transfection of this construct resulted in high-level production of infectious LGTV, which exhibited robust replication kinetics, though slightly slower growth compared to a patient-derived isolate. We further engineered mCherry and Gaussia luciferase reporter versions of the clone, which yielded viruses expressing high levels of their respective reporters while retaining efficient replication. These LGTV infectious clones provide versatile tools for investigating viral replication, gene function, and pathogenesis, and may facilitate screening for antiviral inhibitors. HighlightsO_LISingle-plasmid CMV-driven system launches infectious Langat virus (LGTV) C_LIO_LIThree introns stabilize full-length LGTV cDNA in E. coli C_LIO_LIDNA transfection yields high-titer infectious virus in mammalian cells C_LIO_LIReporter LGTVs enable fluorescent and luminescent infection readouts C_LIO_LIProvides a versatile platform for LGTV genetics and pathogenesis studies C_LI

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Hermacinski, A. I., Richardson, R. B., Bednarski, E., Salim, M., Garg, A., Lim, J. K., Evans, M. J.. 2025-11-13. Construction of a CMV promoter-driven Langat virus cDNA clone and reporter viruses. https://doi.org/10.1101/2025.11.13.688285

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